PROJECT SUMMARY/ABSTRACT Several groups, including ours, have initiated efforts to develop small-animal irradiators that mimic radiation therapy (RT) for human treatment. The major image modality used to guide irradiation is cone-beam CT (CBCT), and our CBCT-small animal radiation research platform (SARRP) was commercialized in 2010. Our system, and others, were transformative for pre-clinical RT research, and 115 machines are now in use world-wide by some 600 investigators. While CBCT provides excellent guidance capability, it is less adept at localizing soft tissue targets growing in a low image contrast environment. In contrast, bioluminescence imaging (BLI), provides strong image contrast and thus is an attractive solution for soft tissue targeting. However, commonly used 2D BLI on an animal surface is inadequate to guide irradiation, because optical transport from an internal bioluminescent tumor is highly susceptible to the effects of irregular torso and tissue optical properties. Recognition of these limitations led us to integrate 3D bioluminescence tomography (BLT) with SARRP. Our first BLT was designed to localize the center of mass (CoM) of an optical target for irradiation. This advance was received with much intrigue, however there was little practical adoption of the BLT system by SARRP users. It was clear that the investigators required two key unmet needs to be addressed, to significantly enhance their conduct of research: 1) knowledge of target shape is a fundamental need for RT. Without such information to guide radiation, large portions of normal tissue can be irradiated unnecessarily, leading to undesired experimental uncertainties. It is imperative that we advance BLT guidance beyond CoM, to a new and precise level of 3D target shape delineation; and 2) clinical practice recognizes the importance of complementary use of functional and anatomical image for RT. BLI measures cellular viability, thus it is an ideal imaging modality for longitudinally monitoring treatment outcome. However, the quantitative information that surface BLI provides for assessment is currently limited or even inaccurate. With the novel reconstruction algorithm and calibration methods proposed in this application, we will establish a new quantitative BLT (QBLT) to address this need. We hypothesize that the QBLT/CBCT-guided small animal radiation system will provide investigators new capabilities to localize soft tissue target, define its shape for conformal irradiation, and non-invasively quantify treatment outcome. Our aims are: Aim 1: design and construct a standalone QBLT system readily adapted to commercial radiation platform; Aim 2: optimize input data, and develop calibration method and reconstruction algorithm for target shape delineation and quantitative imaging; Aim 3: validate the QBLT-guided RT in vivo and assess its suitability for treatment assessment. The success of this proposal will significantly enhance small animal radiotherapy research with the capabilities of functional targeting and assessment beyond anatomical imaging.